Biol. Chem. 2014; 395(7-8): 905–911
Short Communication Anja Menzela, Piotr Neumannb, Christian Schwieger and Milton T. Stubbs*
Thermodynamic signatures in macromolecular interactions involving conformational flexibility Abstract: The energetics of macromolecular interactions are complex, particularly where protein flexibility is involved. Exploiting serendipitous differences in the plasticity of a series of closely related trypsin variants, we analyzed the enthalpic and entropic contributions accompanying interaction with L45K-eglin C. Binding of the four variants show significant differences in released heat, although the affinities vary little, in accordance with the principle of enthalpy-entropy compensation. Binding of the most disordered variant is almost entirely enthalpically driven, with practically no entropy change. As structures of the complexes reveal negligible differences in protein-inhibitor contacts, we conclude that solvent effects contribute significantly to binding affinities. Keywords: crystal structure; enthalpy entropy compensation; isothermal titration calorimetry; protein flexibility; thermodynamics. DOI 10.1515/hsz-2014-0177 Received March 27, 2014; accepted May 5, 2014 Dedicated to Professor Dr. Gerhard Klebe on the occasion of his 60th birthday.
The strength of a macromolecular interaction is commonly quantified according to the equilibrium binding (or affinity) constant, KA ( = 1/KD, where KD is the dissociation a Present address: Institut für Rechtsmedizin, Otto-von-GuerikeUniversität, Leipziger Straße 44, D-39120 Magdeburg, Germany b Present address: Institut für Mikrobiologie und Genetik, Georg-AugustUniversität, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany *Corresponding author: Milton T. Stubbs, Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle/Saale, Germany, e-mail: [email protected] Anja Menzel and Piotr Neumann: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, KurtMothes-Straße 3, D-06120 Halle/Saale, Germany Christian Schwieger: Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, von-Dankelman-Platz 4, D-06120 Halle/Saale, Germany
constant). Thermodynamically, the binding constant represents a standard free energy of binding ∆Gbo = -RTln(K A ) = ∆H bo -T ∆Sbo , where ∆H bo and ∆Sbo are the changes in enthalpy and entropy upon binding, respectively. Isothermal titration calorimetry (ITC) allows direct measurement of the heat associated with binding (or enthalpy change ∆H bo at constant pressure) and the binding constant KA, from which ∆Gbo and ∆Sbo can be derived. The technique has therefore become an invaluable tool for the biophysical characterization of diverse biological systems (Ghai et al., 2012) and has attracted particular interest in drug discovery (Ladbury et al., 2010). A commonly described phenomenon in studies of protein-ligand interactions is that of enthalpy-entropy compensation (Chodera and Mobley, 2013): in a series of related ligands, favorable increases in enthalpic contributions tend to be offset by unfavorable entropic effects, resulting in a little or no change in affinity. These counterbalanced effects may be attributable to changes in solvent structure (e.g., Breiten et al., 2013) and/or protein flexibility (e.g., Cho et al., 2010) that accompany the binding process itself. There is, however, a general lack of suitable model systems to gain a better understanding of these effects. To investigate issues of affinity and selectivity, we have developed a model system in which the ligand binding pocket of coagulation factor Xa, an attractive target for anticoagulant therapy (Straub et al., 2011), has been grafted on to the homologous digestive enzyme trypsin (Reyda et al., 2003; Rauh et al., 2004; see accompanying paper by Tziridis et al., 2014). Unexpectedly, the chimeric factor Xa-like trypsin variants exhibit a range of dynamic properties that allow investigation of the influence of protein flexibility on ligand interactions (Figure 1). Briefly, exchange of three amino acid segments around the ligand binding site of trypsin (Asn97Glu-Thr98-Leu99Tyr, Ser190Ala, and Tyr172Ser-Pro173Ser-Gly174PheGln175Ile) yielded the prototype factor Xa-like trypsin variant TripleSer217Val227, whose structure diverged
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906 A. Menzel et al.: Thermodynamic signatures involving conformational flexibility
Figure 1 Schematic representations of the factor Xa-like trypsin variants analyzed in this paper and their properties. (A) In variant TripleSer217Val227, the side chain of Phe174 and the intermediate helix (IH) are found in a ‘down’ conformation that differs from that of both parent proteinases trypsin and factor Xa (Reyda et al., 2003; Rauh et al., 2004). (B) Mutation of Ser217 to Glu results in the variant TripleGlu217Val227, which adopts the ‘down’ conformation in the presence of low affinity inhibitors but can adopt the factor Xa-like ‘up’ conformation in the presence of high affinity factor Xa inhibitors, with formation of an aromatic box delineated by the side chains of Tyr99, Trp215, and Phe174 (Rauh et al., 2004; Tziridis et al., 2014). (C) Replacement of Val227 by Phe disrupts the ‘down’ conformation, resulting in the destabilized variant TripleSer217Phe227. (D) A combination of the ‘down’ destabilizing Phe227 and the ‘up’ facilitator Glu217 results in variant TripleGlu217Phe227, which exhibits the factor Xa-like ‘up’ conformation only. See also the Supplementary Movie 1 (Tziridis et al., 2014).
significantly from that of both parent proteinases (Reyda et al., 2003; Rauh et al., 2004): the so-called intermediate helix was partially unwound and exhibited a different orientation with respect to the remainder of the protein (the ‘down’ conformation). In particular, mutated residue Phe174 (which, together with Trp215 and Tyr99, forms a distinctive aromatic box in factor Xa) was found buried in the core of the chimeric proteinase. Replacement of Ser217 at the edge of the specificity pocket by the corresponding glutamic acid in factor Xa (TripleGlu217Val227) also resulted in a variant that demonstrated the ‘down’ conformation but which could adopt the factor Xa-like ‘up’ conformation (in which the aromatic box is fully formed) upon binding strong factor Xa ligands (Rauh
et al., 2004). Hydrophobic core residue Val227, which juxtaposes Phe174 in the ‘down’ conformation, was replaced by the bulky Phe227 in an attempt to destabilize the ‘down’ conformation and thereby force the ‘up’ conformation (Tziridis et al., 2014): the resulting variant TripleSer217Phe227 exhibited significant disorder in the absence of factor Xa ligands, however. Finally, combination of both mutations (Ser217Glu and Val227Phe) yielded the variant proteinase TripleGlu217Phe227 that consistently displayed the ‘up’ conformation observed in all factor Xa structures investigated to date. Although much attention has been focused on synthetic small molecule inhibitors of factor Xa, protein inhibitors have also attracted interest, in particular those from hematophagous organisms (Koh and Kini, 2009), which can target factor Xa in the complex with its physiological cofactor factor Va (the prothrombinase complex). With a view to understanding mechanisms of proteinprotein interaction, we have chosen to exploit the features of our chimeric proteinases in a combined ITC and structural study of their interactions with a variant of the leech-derived serine proteinase inhibitor eglin C (Seemuller et al., 1977, 1980). This so-called standard mechanism protein inhibitor (Laskowski and Qasim, 2000) binds to serine proteinases with an extended reactive site loop in a substrate-like or canonical manner (McPhalen et al., 1985; Bolognesi et al., 1990; Bode and Huber, 1991). In contrast to many other naturally occurring serine proteinase inhibitors, the 70 amino acid protein contains no disulfide bonds and is stabilized instead by salt bridges between the protein core and the reactive site loop (Hipler et al., 1992; Hyberts et al., 1992), facilitating soluble recombinant protein production in Escherichia coli. Wild-type eglin C is a potent inhibitor of human leukocyte elastase, cathepsin G, α-chymotrypsin, and subtilisin, but not of trypsin (Seemuller et al., 1980; Heinz et al., 1992). Accordingly, the present studies were carried out using the variant L45Keglin C, in which the wild-type LeuI45 that determines the primary specificity is exchanged for Lys (and where the prefix ‘I’ is used to denote residue numbers of the inhibitor). In addition, the active site Ser195 of all trypsin variants was mutated to Ala to prevent proteolysis, resulting in the variants TripleSer217Val227†, TripleGlu217Val227†, TripleSer217Phe227†, and TripleGlu217Phe227†, where the superscript † denotes an inactive variant. Analyses of the binding isotherms indicate similar affinities of L45K-eglin C for each of the variants under the applied conditions (Figure 2), ranging from KD values of 47 nm (TripleSer217Val227†) to 140 nm (TripleGlu217Phe227†) or standard free energies of binding ∆Gbo between -10.0 and -9.4 kcal/mol, respectively. Meanwhile, significant
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A. Menzel et al.: Thermodynamic signatures involving conformational flexibility 907
Figure 2 Interactions of the inactive (Ser195Ala) trypsin† variants with L45K-eglin C measured by ITC. Binding of L45K-eglin C (syringe) to (A) TripleSer217Val227†, (B) TripleGlu217Val227†, (C) TripleSer217Phe227†, and (D) TripleGlu217Phe227† (measurement cell) is shown. Upper panels: raw titration data; lower panels: integrated enthalpies. Data were corrected for dilution heat and fitted to a single-site binding model (solid line). The commonly used unit calorie (cal) can be converted into the SI unit joule (J) using 1 J = 4.182 cal. Methods: For the thermodynamic studies described here, it was necessary to use proteolytically inactive variants. To this end, the active site Ser195 each of the variants was mutated to Ala using the Quick-Change™ kit (Weiner et al., 1994), and the primers 5′ CAC AGG GCC ACC TGC GTC ACC CTG GC 3′ (forward) and 5′ GC CAG GGT GAC GCA GGT GGC CCT GTG 3′ (reverse). The inactive trypsin variants (denoted in this paper by the superscript †) were expressed as inclusion bodies in E. coli, refolded and purified as described previously (Rauh et al., 2002, 2004; Tziridis et al., 2014). Production of L45K-eglin C is described in the supplementary information. Preliminary ITC experiments with TripleSer217Val227† and L45K-eglin C using a 20-mm HEPES/NaOH buffer at pH 7.5 with 1 mm CaCl2 revealed a sub-nanomolar KD (data not shown); this was increased by lowering the pH to enter the effective range of the ITC experiment. All proteins were dialyzed against 50 mm MES/NaOH, 125 mm NaCl, 1 mm CaCl2, pH 6.0, prior to ITC measurements at 25°C using a MicroCal VP-ITC titration microcalorimeter (MicroCal, Northampton, MA, USA). A series of 1-μl injections of L45K-eglin C (70–100 μm) were added sequentially to the inactive trypsin variants, which were in the cell at a concentration of ca. 100*KD. Heats of dilution from injecting L45K-eglin C into measurement buffer were subtracted before curve fitting. Values for each thermodynamic parameter ( ∆Hbo , KD, ∆Gbo , and T ∆Sbo , insets) are based on at least two independent titrations.
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908 A. Menzel et al.: Thermodynamic signatures involving conformational flexibility differences are observed in the released heats on binding: variant TripleSer217Phe227† showed the largest exothermic interaction ( ∆H bo = -8.4 kcal/mol), followed by TripleSer217Val227† ( ∆H bo = -6.3 kcal/mol), TripleGlu217Phe227† ( ∆H bo = -4.5 kcal/mol), and TripleGlu217Val227† ( ∆H bo = -3.9 kcal/mol). As the affinities are so similar, these differences in enthalpy must be compensated by entropic effects. Accordingly, the determined entropy changes are different, but in each case positive (-T ∆Sbo < 0 ), indicating that inhibitor binding is driven by favorable changes in both enthalpy and entropy. Each of the variants could be crystallized in complex with L45K-eglin C (Figure 3). All four variant complexes crystallized isomorphically in the monoclinic crystal form
P21 with two complexes A and B in the asymmetric unit. The intermediate helix region of chain A juxtaposes the C-terminal helix of a symmetry related molecule, whereas the corresponding region in chain B is largely exposed to solvent. In each of the eight complexes, L45K-eglin C binds to the variant proteinase in the canonical binding mode (Bode and Huber, 1991; Laskowski and Qasim, 2000), with the primary specificity pocket occupied by residue LeuI45Lys of the inhibitor (Supplementary Figure 1). Three regions of the inhibitor contact the proteinase: the N-terminal residues ThrI1 to LeuI7 and the three C-terminal amino acids HisI68-GlyI70 interact with the autolysis loop Thr149-Asp153, whereas the reactive site loop residues GlyI40-ArgI48 bind in a substrate-like manner
Figure 3 Superposition of crystal structures of the variants with bound L45K-eglin C (yellow sticks) in the region of the intermediate helix (stereo representation). With the exception of TripleSer217Phe227† (red/pink), only minimal differences are seen between the independent chains A and B in the asymmetric unit. TripleSer217Val227† (gray) and TripleGlu217Val227† (blue) are both found in the ‘down’ conformation, whereas TripleGlu217Phe227† adopts the ‘up’ conformation. Although density for residues Ser173-Phe174-Ile175 of TripleSer217Phe227† chain B is weak, it adopts a ‘near-down’ conformation (red); in the same crystal, Phe174 of chain A (pink) loops away from the enzyme, a conformation that is strongly affected by crystal contacts. Methods: Each trypsin variant (in 50 mm MES-NaOH, pH 6.0, 125 mm NaCl, 1 mm CaCl2) was mixed in 1:10 molar stoichiometry with L45Keglin C in the same buffer and incubated at room temperature for 20 min. The complex was purified by size exclusion chromatography using a HiLoad-26/60-Superdex 75 column (GE Healthcare), re-buffered to 50 mm MES-NaOH, pH 6.0, 20 mm NaCl, 1 mm CaCl2, concentrated to 4.5 mg/ml, and centrifuged for 10 min at 20 000 g. Crystallization screening for the TripleGlu217Val227†-L45K-eglin C complex in sitting drops (200 nl protein solution/200 nl crystallization solution) at 13°C yielded diffraction quality crystals using the condition D1 of the JBS Classic 5 Screen (Jena Bioscience, Jena, Germany) (18% w/v PEG 10 000, 20% v/v glycerol, 100 mm Tris-HCl, pH 8.5, and 100 mm NaCl). The remaining three complexes were crystallized under similar conditions using the hanging-drop method (2 μl of protein solution/2 μl of crystallization solution equilibrated against 500 μl reservoir solution). The TripleGlu217Phe227†-L45K-eglin C complex was crystallized within 6 days from the same crystallization buffer, albeit at a pH of 9.0. Well diffracting crystals of the complexes TripleSer217Val227†-L45K-eglin C and TripleSer217Phe227†-L45K-eglin C were only obtained by cross-seeding using a cat whisker and a crushed crystal of TripleGlu217Phe227†L45K-eglin C. Within 7 days, crystals large enough for X-ray diffraction studies grew from the seeded drops at pH 8.5. Crystals were shock cooled in a nitrogen stream for data collection without further cryoprotectant and diffracted X-rays beyond 2.0-Å resolution. Data were collected at beam lines X12 (EMBL, DESY, Hamburg, Germany) and BL14.1 (BESSY, HZB Berlin), both equipped with a MAR-Mosaic 225-mm CCD detector (Rayonix, Evanston, IL, USA) and processed using XDS/XSCALE (Kabsch, 2010) and MOSFLM/SCALA (Leslie, 2006; Winn et al., 2011). The structure of TripleSer217Val227†-L45K-eglin C was solved by molecular replacement using Phaser (McCoy et al., 2007) [search model coordinates 1v2l (Rauh et al., 2004) (TripleSer217Val227) and 1acb (Frigerio et al., 1992) (native-eglin C)] followed by cycles of manual rebuilding using Coot (Emsley and Cowtan, 2004) and refinement using Refmac (Murshudov et al., 1997). The remaining isomorphous structures were solved using the TripleSer217Val227†-L45K-eglin C coordinates as start model. All data collection and refinement statistics are given in Supplementary Table 1.
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A. Menzel et al.: Thermodynamic signatures involving conformational flexibility 909
to the proteinase subsites S6-S3′. Interactions within the complexes are on the whole indistinguishable, with buried accessible surface areas between 850 and 975 Å2 (Krissinel and Henrick, 2007) – the small differences are for the most part attributable to variable ordering of the N-terminal inhibitor residues (ThrI1-GlyI5), which in turn correlate with minor changes in the crystal packing (reflected in marginal differences in the cell constants b, c, and β). Owing to the presence of the non-glycyl P2 residue (ThrI44), the side chain of Tyr99 rotates around 30° in χ2 as observed in the kallikrein:BPTI complex (Chen and Bode, 1983) and proposed for the factor Xa:tissue factor pathway inhibitor complex (Burgering et al., 1997). The only obvious differences between the complexes are observed in the orientations of Phe174 and the intermediate helix (Figure 3). In the complexes with TripleSer217Val227† and TripleGlu217Val227†, each proteinase monomer in the asymmetric unit is found in the ‘down’ conformation. Similarly, both molecules of TripleGlu217Phe227† are found in the ‘up’ conformation, although the side chain of Phe174 rotates away from its factor Xa-like aromatic box position to avoid clashes with the P4 residue Pro42I, which nestles in a small induced pocket formed by the side chains of Tyr99, Phe174, Trp215, and Glu217. Meanwhile, two conformations are observed for TripleSer217Phe227†: a partially disordered ‘near-down’ conformation in chain B1 and a ‘looped-out’ structure in chain A in which the side chain of Phe174 is surface exposed in a symmetry-stabilized conformation. We note that a number of ordered solvent molecules are located near to each copy of the Phe174 loop region; in each case, however, their positions are influenced by crystal packing, precluding any meaningful analysis. We can draw three main conclusions from the structural data. First, both variants TripleSer217Val227† and TripleGlu217Val227† bind L45K-eglin C in the ‘down’ conformation (even though the ‘up’ conformation is allowed in the crystal form). Second, as two conformations are observed for the variant TripleSer217Phe227† that depend on the crystallographic environment, we infer that this variant can maintain a disordered conformation, even in a complex with the inhibitor L45K-eglin C. Finally, the variant TripleGlu217Phe227† binds L45K-eglin C in the ‘up’ conformation. In addition, the fact that the variant 1 The disorder is reflected in higher temperature factors of the final model in this region; a more detailed analysis of these has not been carried out because the link between crystallographic B factors and dynamics is questionable, demonstrated recently for a set of representative proteins (Reichert et al., 2012)
TripleGlu217Val227† is found in the ‘down’ conformation suggests that binding of L45K-eglin C to the ‘up’ conformation may be energetically disfavored, perhaps due to reorientation of the Phe174 side chain. How can these structural data be reconciled with the calorimetric data? The fact that TripleSer217Phe227† maintains significant degrees of freedom upon complex formation is in agreement with the minimal entropy change observed ( -T ∆Sbo = -1.3 kcal/mol). Remarkably, the interaction of TripleSer217Val227† with L45K-eglin C shows a less negative enthalpy relative to TripleSer217Phe227† ( ∆∆H bo ≈ 2 kcal/mol), even though the proteinase-inhibitor interactions are equivalent in the two crystals. We therefore surmise that this difference in enthalpy change must be due to the breaking of favorable bonds involving solvent molecules upon TripleSer217Val227†:L45Keglin C complex formation. Concomitantly, the breaking of solvent bonds leads to an increase in degrees of freedom of the solvent molecules, consistent with an increase in the entropic contribution to the ligand binding ( -T ∆∆Sbo = -2.3 kcal/mol). Consequently, this suggests that disorder in the variant TripleSer217Phe227† is accompanied by a corresponding disordered solvent shell, allowing the protein to adapt to ligand binding without losing contacts to hydration water. Binding of the inhibitor in this case requires neither the breaking of bonds to ordered solvent in this region, nor a change in degrees of freedom of either the protein or the solvent. In contrast, the two variants TripleSer217Val227† and TripleGlu217Val227† display the same conformations in their complexes with L45K-eglin C, yet a slight drop in affinity is observed for the latter that is accompanied by a decrease in negative enthalpy ( ∆∆H bo ≈ 2.5 kcal/mol) and an increased entropic contribution ( -T ∆∆Sbo ≈ -1.9 kcal/mol). This appears to be counterintuitive: one would expect fixation of TripleGlu217Val227† in the ‘down’ conformation upon complex formation to lead to a reduction of conformational degrees of freedom of the trypsin variant and therefore an entropic penalty. One possible explanation could be that introduction of the polar and negatively charged Glu217 results in increased solvation of this variant, so that inhibitor binding leads to displacement of ordered solvent molecules, a loss in enthalpy, and a concomitant gain in entropy. Finally, although variant TripleGlu217Phe227† binds to L45K-eglin C in the ‘up’ conformation, its thermodynamic signature is very similar to that of TripleGlu217Val227†. This implies that in this case, the proteinase conformation is of minor importance for the thermodynamics of binding. Rather, it seems likely that the substitution of Ser217 for the charged Glu has an important impact on
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910 A. Menzel et al.: Thermodynamic signatures involving conformational flexibility enthalpy and entropy changes upon inhibitor binding. It should, however, be borne in mind that macromolecular complex formation involves the summation of a myriad of interactions that energetically can be both favorable and unfavorable, precluding a precise atomic level description of the binding isotherms. In conclusion, it appears that differing extents of solvent reorganization provide an explanation for the encountered enthalpy-entropy compensation observed upon inhibitor binding to the different trypsin variants. The comparable magnitudes of the breaking of ordered solvent hydrogen bonds (resulting in enthalpy loss of the system) and the simultaneous increase in solvent mobility (leading to entropy gain) counterbalance each other energetically, resulting in a near constant free energy of binding – even though the thermodynamic signatures (the driving forces behind the interactions) are fundamentally different. The results presented here highlight the difficulties in correlating thermodynamic data with structural data. In many cases, order-disorder changes of the target protein of the kind described here are difficult to detect or analyze. Moreover, we note that changes in solvent orderdisorder can contribute significantly to both enthalpy and entropy changes. Observation of such changes is generally not accessible through X-ray crystallography: disordered solvent molecules are, by definition, undetectable in crystal structures, whereas ordered solvents in crystals are, in practice, only observed within the immediate vicinity of the macromolecular entity and are strongly affected by crystal contacts. Our results emphasize the fact that the interpretation of ITC data demands a description of the entire system (Ladbury, 2010), including solvent molecules and conformational changes. The use of well-defined model systems such as that described here will aid understanding of the forces driving proteinprotein interactions and provide a basis for influencing them. Acknowledgments: This work was supported by the DFG Graduiertenkolleg 1026 ‘Conformational transitions in macromolecular interactions’.
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Short Communication Anja Menzela, Piotr Neumannb, Christian Schwieger and Milton T. Stubbs*
Thermodynamic signatures in macromolecular interactions involving conformational flexibility Abstract: The energetics of macromolecular interactions are complex, particularly where protein flexibility is involved. Exploiting serendipitous differences in the plasticity of a series of closely related trypsin variants, we analyzed the enthalpic and entropic contributions accompanying interaction with L45K-eglin C. Binding of the four variants show significant differences in released heat, although the affinities vary little, in accordance with the principle of enthalpy-entropy compensation. Binding of the most disordered variant is almost entirely enthalpically driven, with practically no entropy change. As structures of the complexes reveal negligible differences in protein-inhibitor contacts, we conclude that solvent effects contribute significantly to binding affinities. Keywords: crystal structure; enthalpy entropy compensation; isothermal titration calorimetry; protein flexibility; thermodynamics. DOI 10.1515/hsz-2014-0177 Received March 27, 2014; accepted May 5, 2014 Dedicated to Professor Dr. Gerhard Klebe on the occasion of his 60th birthday.
The strength of a macromolecular interaction is commonly quantified according to the equilibrium binding (or affinity) constant, KA ( = 1/KD, where KD is the dissociation a Present address: Institut für Rechtsmedizin, Otto-von-GuerikeUniversität, Leipziger Straße 44, D-39120 Magdeburg, Germany b Present address: Institut für Mikrobiologie und Genetik, Georg-AugustUniversität, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany *Corresponding author: Milton T. Stubbs, Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle/Saale, Germany, e-mail: [email protected] Anja Menzel and Piotr Neumann: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, KurtMothes-Straße 3, D-06120 Halle/Saale, Germany Christian Schwieger: Institut für Chemie, Martin-Luther-Universität Halle-Wittenberg, von-Dankelman-Platz 4, D-06120 Halle/Saale, Germany
constant). Thermodynamically, the binding constant represents a standard free energy of binding ∆Gbo = -RTln(K A ) = ∆H bo -T ∆Sbo , where ∆H bo and ∆Sbo are the changes in enthalpy and entropy upon binding, respectively. Isothermal titration calorimetry (ITC) allows direct measurement of the heat associated with binding (or enthalpy change ∆H bo at constant pressure) and the binding constant KA, from which ∆Gbo and ∆Sbo can be derived. The technique has therefore become an invaluable tool for the biophysical characterization of diverse biological systems (Ghai et al., 2012) and has attracted particular interest in drug discovery (Ladbury et al., 2010). A commonly described phenomenon in studies of protein-ligand interactions is that of enthalpy-entropy compensation (Chodera and Mobley, 2013): in a series of related ligands, favorable increases in enthalpic contributions tend to be offset by unfavorable entropic effects, resulting in a little or no change in affinity. These counterbalanced effects may be attributable to changes in solvent structure (e.g., Breiten et al., 2013) and/or protein flexibility (e.g., Cho et al., 2010) that accompany the binding process itself. There is, however, a general lack of suitable model systems to gain a better understanding of these effects. To investigate issues of affinity and selectivity, we have developed a model system in which the ligand binding pocket of coagulation factor Xa, an attractive target for anticoagulant therapy (Straub et al., 2011), has been grafted on to the homologous digestive enzyme trypsin (Reyda et al., 2003; Rauh et al., 2004; see accompanying paper by Tziridis et al., 2014). Unexpectedly, the chimeric factor Xa-like trypsin variants exhibit a range of dynamic properties that allow investigation of the influence of protein flexibility on ligand interactions (Figure 1). Briefly, exchange of three amino acid segments around the ligand binding site of trypsin (Asn97Glu-Thr98-Leu99Tyr, Ser190Ala, and Tyr172Ser-Pro173Ser-Gly174PheGln175Ile) yielded the prototype factor Xa-like trypsin variant TripleSer217Val227, whose structure diverged
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906 A. Menzel et al.: Thermodynamic signatures involving conformational flexibility
Figure 1 Schematic representations of the factor Xa-like trypsin variants analyzed in this paper and their properties. (A) In variant TripleSer217Val227, the side chain of Phe174 and the intermediate helix (IH) are found in a ‘down’ conformation that differs from that of both parent proteinases trypsin and factor Xa (Reyda et al., 2003; Rauh et al., 2004). (B) Mutation of Ser217 to Glu results in the variant TripleGlu217Val227, which adopts the ‘down’ conformation in the presence of low affinity inhibitors but can adopt the factor Xa-like ‘up’ conformation in the presence of high affinity factor Xa inhibitors, with formation of an aromatic box delineated by the side chains of Tyr99, Trp215, and Phe174 (Rauh et al., 2004; Tziridis et al., 2014). (C) Replacement of Val227 by Phe disrupts the ‘down’ conformation, resulting in the destabilized variant TripleSer217Phe227. (D) A combination of the ‘down’ destabilizing Phe227 and the ‘up’ facilitator Glu217 results in variant TripleGlu217Phe227, which exhibits the factor Xa-like ‘up’ conformation only. See also the Supplementary Movie 1 (Tziridis et al., 2014).
significantly from that of both parent proteinases (Reyda et al., 2003; Rauh et al., 2004): the so-called intermediate helix was partially unwound and exhibited a different orientation with respect to the remainder of the protein (the ‘down’ conformation). In particular, mutated residue Phe174 (which, together with Trp215 and Tyr99, forms a distinctive aromatic box in factor Xa) was found buried in the core of the chimeric proteinase. Replacement of Ser217 at the edge of the specificity pocket by the corresponding glutamic acid in factor Xa (TripleGlu217Val227) also resulted in a variant that demonstrated the ‘down’ conformation but which could adopt the factor Xa-like ‘up’ conformation (in which the aromatic box is fully formed) upon binding strong factor Xa ligands (Rauh
et al., 2004). Hydrophobic core residue Val227, which juxtaposes Phe174 in the ‘down’ conformation, was replaced by the bulky Phe227 in an attempt to destabilize the ‘down’ conformation and thereby force the ‘up’ conformation (Tziridis et al., 2014): the resulting variant TripleSer217Phe227 exhibited significant disorder in the absence of factor Xa ligands, however. Finally, combination of both mutations (Ser217Glu and Val227Phe) yielded the variant proteinase TripleGlu217Phe227 that consistently displayed the ‘up’ conformation observed in all factor Xa structures investigated to date. Although much attention has been focused on synthetic small molecule inhibitors of factor Xa, protein inhibitors have also attracted interest, in particular those from hematophagous organisms (Koh and Kini, 2009), which can target factor Xa in the complex with its physiological cofactor factor Va (the prothrombinase complex). With a view to understanding mechanisms of proteinprotein interaction, we have chosen to exploit the features of our chimeric proteinases in a combined ITC and structural study of their interactions with a variant of the leech-derived serine proteinase inhibitor eglin C (Seemuller et al., 1977, 1980). This so-called standard mechanism protein inhibitor (Laskowski and Qasim, 2000) binds to serine proteinases with an extended reactive site loop in a substrate-like or canonical manner (McPhalen et al., 1985; Bolognesi et al., 1990; Bode and Huber, 1991). In contrast to many other naturally occurring serine proteinase inhibitors, the 70 amino acid protein contains no disulfide bonds and is stabilized instead by salt bridges between the protein core and the reactive site loop (Hipler et al., 1992; Hyberts et al., 1992), facilitating soluble recombinant protein production in Escherichia coli. Wild-type eglin C is a potent inhibitor of human leukocyte elastase, cathepsin G, α-chymotrypsin, and subtilisin, but not of trypsin (Seemuller et al., 1980; Heinz et al., 1992). Accordingly, the present studies were carried out using the variant L45Keglin C, in which the wild-type LeuI45 that determines the primary specificity is exchanged for Lys (and where the prefix ‘I’ is used to denote residue numbers of the inhibitor). In addition, the active site Ser195 of all trypsin variants was mutated to Ala to prevent proteolysis, resulting in the variants TripleSer217Val227†, TripleGlu217Val227†, TripleSer217Phe227†, and TripleGlu217Phe227†, where the superscript † denotes an inactive variant. Analyses of the binding isotherms indicate similar affinities of L45K-eglin C for each of the variants under the applied conditions (Figure 2), ranging from KD values of 47 nm (TripleSer217Val227†) to 140 nm (TripleGlu217Phe227†) or standard free energies of binding ∆Gbo between -10.0 and -9.4 kcal/mol, respectively. Meanwhile, significant
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A. Menzel et al.: Thermodynamic signatures involving conformational flexibility 907
Figure 2 Interactions of the inactive (Ser195Ala) trypsin† variants with L45K-eglin C measured by ITC. Binding of L45K-eglin C (syringe) to (A) TripleSer217Val227†, (B) TripleGlu217Val227†, (C) TripleSer217Phe227†, and (D) TripleGlu217Phe227† (measurement cell) is shown. Upper panels: raw titration data; lower panels: integrated enthalpies. Data were corrected for dilution heat and fitted to a single-site binding model (solid line). The commonly used unit calorie (cal) can be converted into the SI unit joule (J) using 1 J = 4.182 cal. Methods: For the thermodynamic studies described here, it was necessary to use proteolytically inactive variants. To this end, the active site Ser195 each of the variants was mutated to Ala using the Quick-Change™ kit (Weiner et al., 1994), and the primers 5′ CAC AGG GCC ACC TGC GTC ACC CTG GC 3′ (forward) and 5′ GC CAG GGT GAC GCA GGT GGC CCT GTG 3′ (reverse). The inactive trypsin variants (denoted in this paper by the superscript †) were expressed as inclusion bodies in E. coli, refolded and purified as described previously (Rauh et al., 2002, 2004; Tziridis et al., 2014). Production of L45K-eglin C is described in the supplementary information. Preliminary ITC experiments with TripleSer217Val227† and L45K-eglin C using a 20-mm HEPES/NaOH buffer at pH 7.5 with 1 mm CaCl2 revealed a sub-nanomolar KD (data not shown); this was increased by lowering the pH to enter the effective range of the ITC experiment. All proteins were dialyzed against 50 mm MES/NaOH, 125 mm NaCl, 1 mm CaCl2, pH 6.0, prior to ITC measurements at 25°C using a MicroCal VP-ITC titration microcalorimeter (MicroCal, Northampton, MA, USA). A series of 1-μl injections of L45K-eglin C (70–100 μm) were added sequentially to the inactive trypsin variants, which were in the cell at a concentration of ca. 100*KD. Heats of dilution from injecting L45K-eglin C into measurement buffer were subtracted before curve fitting. Values for each thermodynamic parameter ( ∆Hbo , KD, ∆Gbo , and T ∆Sbo , insets) are based on at least two independent titrations.
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908 A. Menzel et al.: Thermodynamic signatures involving conformational flexibility differences are observed in the released heats on binding: variant TripleSer217Phe227† showed the largest exothermic interaction ( ∆H bo = -8.4 kcal/mol), followed by TripleSer217Val227† ( ∆H bo = -6.3 kcal/mol), TripleGlu217Phe227† ( ∆H bo = -4.5 kcal/mol), and TripleGlu217Val227† ( ∆H bo = -3.9 kcal/mol). As the affinities are so similar, these differences in enthalpy must be compensated by entropic effects. Accordingly, the determined entropy changes are different, but in each case positive (-T ∆Sbo < 0 ), indicating that inhibitor binding is driven by favorable changes in both enthalpy and entropy. Each of the variants could be crystallized in complex with L45K-eglin C (Figure 3). All four variant complexes crystallized isomorphically in the monoclinic crystal form
P21 with two complexes A and B in the asymmetric unit. The intermediate helix region of chain A juxtaposes the C-terminal helix of a symmetry related molecule, whereas the corresponding region in chain B is largely exposed to solvent. In each of the eight complexes, L45K-eglin C binds to the variant proteinase in the canonical binding mode (Bode and Huber, 1991; Laskowski and Qasim, 2000), with the primary specificity pocket occupied by residue LeuI45Lys of the inhibitor (Supplementary Figure 1). Three regions of the inhibitor contact the proteinase: the N-terminal residues ThrI1 to LeuI7 and the three C-terminal amino acids HisI68-GlyI70 interact with the autolysis loop Thr149-Asp153, whereas the reactive site loop residues GlyI40-ArgI48 bind in a substrate-like manner
Figure 3 Superposition of crystal structures of the variants with bound L45K-eglin C (yellow sticks) in the region of the intermediate helix (stereo representation). With the exception of TripleSer217Phe227† (red/pink), only minimal differences are seen between the independent chains A and B in the asymmetric unit. TripleSer217Val227† (gray) and TripleGlu217Val227† (blue) are both found in the ‘down’ conformation, whereas TripleGlu217Phe227† adopts the ‘up’ conformation. Although density for residues Ser173-Phe174-Ile175 of TripleSer217Phe227† chain B is weak, it adopts a ‘near-down’ conformation (red); in the same crystal, Phe174 of chain A (pink) loops away from the enzyme, a conformation that is strongly affected by crystal contacts. Methods: Each trypsin variant (in 50 mm MES-NaOH, pH 6.0, 125 mm NaCl, 1 mm CaCl2) was mixed in 1:10 molar stoichiometry with L45Keglin C in the same buffer and incubated at room temperature for 20 min. The complex was purified by size exclusion chromatography using a HiLoad-26/60-Superdex 75 column (GE Healthcare), re-buffered to 50 mm MES-NaOH, pH 6.0, 20 mm NaCl, 1 mm CaCl2, concentrated to 4.5 mg/ml, and centrifuged for 10 min at 20 000 g. Crystallization screening for the TripleGlu217Val227†-L45K-eglin C complex in sitting drops (200 nl protein solution/200 nl crystallization solution) at 13°C yielded diffraction quality crystals using the condition D1 of the JBS Classic 5 Screen (Jena Bioscience, Jena, Germany) (18% w/v PEG 10 000, 20% v/v glycerol, 100 mm Tris-HCl, pH 8.5, and 100 mm NaCl). The remaining three complexes were crystallized under similar conditions using the hanging-drop method (2 μl of protein solution/2 μl of crystallization solution equilibrated against 500 μl reservoir solution). The TripleGlu217Phe227†-L45K-eglin C complex was crystallized within 6 days from the same crystallization buffer, albeit at a pH of 9.0. Well diffracting crystals of the complexes TripleSer217Val227†-L45K-eglin C and TripleSer217Phe227†-L45K-eglin C were only obtained by cross-seeding using a cat whisker and a crushed crystal of TripleGlu217Phe227†L45K-eglin C. Within 7 days, crystals large enough for X-ray diffraction studies grew from the seeded drops at pH 8.5. Crystals were shock cooled in a nitrogen stream for data collection without further cryoprotectant and diffracted X-rays beyond 2.0-Å resolution. Data were collected at beam lines X12 (EMBL, DESY, Hamburg, Germany) and BL14.1 (BESSY, HZB Berlin), both equipped with a MAR-Mosaic 225-mm CCD detector (Rayonix, Evanston, IL, USA) and processed using XDS/XSCALE (Kabsch, 2010) and MOSFLM/SCALA (Leslie, 2006; Winn et al., 2011). The structure of TripleSer217Val227†-L45K-eglin C was solved by molecular replacement using Phaser (McCoy et al., 2007) [search model coordinates 1v2l (Rauh et al., 2004) (TripleSer217Val227) and 1acb (Frigerio et al., 1992) (native-eglin C)] followed by cycles of manual rebuilding using Coot (Emsley and Cowtan, 2004) and refinement using Refmac (Murshudov et al., 1997). The remaining isomorphous structures were solved using the TripleSer217Val227†-L45K-eglin C coordinates as start model. All data collection and refinement statistics are given in Supplementary Table 1.
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A. Menzel et al.: Thermodynamic signatures involving conformational flexibility 909
to the proteinase subsites S6-S3′. Interactions within the complexes are on the whole indistinguishable, with buried accessible surface areas between 850 and 975 Å2 (Krissinel and Henrick, 2007) – the small differences are for the most part attributable to variable ordering of the N-terminal inhibitor residues (ThrI1-GlyI5), which in turn correlate with minor changes in the crystal packing (reflected in marginal differences in the cell constants b, c, and β). Owing to the presence of the non-glycyl P2 residue (ThrI44), the side chain of Tyr99 rotates around 30° in χ2 as observed in the kallikrein:BPTI complex (Chen and Bode, 1983) and proposed for the factor Xa:tissue factor pathway inhibitor complex (Burgering et al., 1997). The only obvious differences between the complexes are observed in the orientations of Phe174 and the intermediate helix (Figure 3). In the complexes with TripleSer217Val227† and TripleGlu217Val227†, each proteinase monomer in the asymmetric unit is found in the ‘down’ conformation. Similarly, both molecules of TripleGlu217Phe227† are found in the ‘up’ conformation, although the side chain of Phe174 rotates away from its factor Xa-like aromatic box position to avoid clashes with the P4 residue Pro42I, which nestles in a small induced pocket formed by the side chains of Tyr99, Phe174, Trp215, and Glu217. Meanwhile, two conformations are observed for TripleSer217Phe227†: a partially disordered ‘near-down’ conformation in chain B1 and a ‘looped-out’ structure in chain A in which the side chain of Phe174 is surface exposed in a symmetry-stabilized conformation. We note that a number of ordered solvent molecules are located near to each copy of the Phe174 loop region; in each case, however, their positions are influenced by crystal packing, precluding any meaningful analysis. We can draw three main conclusions from the structural data. First, both variants TripleSer217Val227† and TripleGlu217Val227† bind L45K-eglin C in the ‘down’ conformation (even though the ‘up’ conformation is allowed in the crystal form). Second, as two conformations are observed for the variant TripleSer217Phe227† that depend on the crystallographic environment, we infer that this variant can maintain a disordered conformation, even in a complex with the inhibitor L45K-eglin C. Finally, the variant TripleGlu217Phe227† binds L45K-eglin C in the ‘up’ conformation. In addition, the fact that the variant 1 The disorder is reflected in higher temperature factors of the final model in this region; a more detailed analysis of these has not been carried out because the link between crystallographic B factors and dynamics is questionable, demonstrated recently for a set of representative proteins (Reichert et al., 2012)
TripleGlu217Val227† is found in the ‘down’ conformation suggests that binding of L45K-eglin C to the ‘up’ conformation may be energetically disfavored, perhaps due to reorientation of the Phe174 side chain. How can these structural data be reconciled with the calorimetric data? The fact that TripleSer217Phe227† maintains significant degrees of freedom upon complex formation is in agreement with the minimal entropy change observed ( -T ∆Sbo = -1.3 kcal/mol). Remarkably, the interaction of TripleSer217Val227† with L45K-eglin C shows a less negative enthalpy relative to TripleSer217Phe227† ( ∆∆H bo ≈ 2 kcal/mol), even though the proteinase-inhibitor interactions are equivalent in the two crystals. We therefore surmise that this difference in enthalpy change must be due to the breaking of favorable bonds involving solvent molecules upon TripleSer217Val227†:L45Keglin C complex formation. Concomitantly, the breaking of solvent bonds leads to an increase in degrees of freedom of the solvent molecules, consistent with an increase in the entropic contribution to the ligand binding ( -T ∆∆Sbo = -2.3 kcal/mol). Consequently, this suggests that disorder in the variant TripleSer217Phe227† is accompanied by a corresponding disordered solvent shell, allowing the protein to adapt to ligand binding without losing contacts to hydration water. Binding of the inhibitor in this case requires neither the breaking of bonds to ordered solvent in this region, nor a change in degrees of freedom of either the protein or the solvent. In contrast, the two variants TripleSer217Val227† and TripleGlu217Val227† display the same conformations in their complexes with L45K-eglin C, yet a slight drop in affinity is observed for the latter that is accompanied by a decrease in negative enthalpy ( ∆∆H bo ≈ 2.5 kcal/mol) and an increased entropic contribution ( -T ∆∆Sbo ≈ -1.9 kcal/mol). This appears to be counterintuitive: one would expect fixation of TripleGlu217Val227† in the ‘down’ conformation upon complex formation to lead to a reduction of conformational degrees of freedom of the trypsin variant and therefore an entropic penalty. One possible explanation could be that introduction of the polar and negatively charged Glu217 results in increased solvation of this variant, so that inhibitor binding leads to displacement of ordered solvent molecules, a loss in enthalpy, and a concomitant gain in entropy. Finally, although variant TripleGlu217Phe227† binds to L45K-eglin C in the ‘up’ conformation, its thermodynamic signature is very similar to that of TripleGlu217Val227†. This implies that in this case, the proteinase conformation is of minor importance for the thermodynamics of binding. Rather, it seems likely that the substitution of Ser217 for the charged Glu has an important impact on
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910 A. Menzel et al.: Thermodynamic signatures involving conformational flexibility enthalpy and entropy changes upon inhibitor binding. It should, however, be borne in mind that macromolecular complex formation involves the summation of a myriad of interactions that energetically can be both favorable and unfavorable, precluding a precise atomic level description of the binding isotherms. In conclusion, it appears that differing extents of solvent reorganization provide an explanation for the encountered enthalpy-entropy compensation observed upon inhibitor binding to the different trypsin variants. The comparable magnitudes of the breaking of ordered solvent hydrogen bonds (resulting in enthalpy loss of the system) and the simultaneous increase in solvent mobility (leading to entropy gain) counterbalance each other energetically, resulting in a near constant free energy of binding – even though the thermodynamic signatures (the driving forces behind the interactions) are fundamentally different. The results presented here highlight the difficulties in correlating thermodynamic data with structural data. In many cases, order-disorder changes of the target protein of the kind described here are difficult to detect or analyze. Moreover, we note that changes in solvent orderdisorder can contribute significantly to both enthalpy and entropy changes. Observation of such changes is generally not accessible through X-ray crystallography: disordered solvent molecules are, by definition, undetectable in crystal structures, whereas ordered solvents in crystals are, in practice, only observed within the immediate vicinity of the macromolecular entity and are strongly affected by crystal contacts. Our results emphasize the fact that the interpretation of ITC data demands a description of the entire system (Ladbury, 2010), including solvent molecules and conformational changes. The use of well-defined model systems such as that described here will aid understanding of the forces driving proteinprotein interactions and provide a basis for influencing them. Acknowledgments: This work was supported by the DFG Graduiertenkolleg 1026 ‘Conformational transitions in macromolecular interactions’.
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